Immunoadhesins (or immunoglobulin fusion proteins) are antibody-like molecules resulting from the fusion of a fragment (e.g., Fc portion) of an immunoglobulin and a ligand-binding region of a receptor or an adhesive molecule. The typical immunoadhesins known in the art have the structure of an antibody in which the variable region, participating in antigen recognition, is replaced with a ligand-binding region of a receptor while retaining the Fc portion. For a long time, a large number of patents have described fusion proteins in which a specific region of a physiologically active protein is linked to an antibody (U.S. Pat. Nos. 5,521,288, 5,844,095, 6,046,310, 6,090,914, 6,100,383 and 6,225,448).
The immunoadhesin has the following advantages over a molecule not containing an immunoglobulin:
1) the fusion protein has increased total avidity to a ligand because it has bivalency in a dimer form;
2) the fusion protein is present in an undestroyed form in serum for a longer period of time by virtue of increased molecular stability;
3) effector cells are activated by the Fc (Fragment crystallizable) portion of the immunoglobulin heavy chain; and
4) the fusion protein is isolated and purified by a convenient method, for example, using protein A.
For example, in the case of tumor necrosis factor (hereinafter, referred to simply as “TNF”) as a cytokine, to suppress TNF-dependent inflammation responses, tumor necrosis factor receptor (hereinafter, referred to simply as “TNFR”) may be used as described in PCT Publication Nos. WO92/16221 and WO95/34326, or a TNFR-immunoglobulin(Ig) fusion protein may be used as described in U.S. Pat. No. 5,447,851 and PCT Publication No. WO94/06476. According to numerous reports, TNFR-Ig fusion proteins have much higher affinity to TNF than the native monomer form or Ig-non-fused form of TNFR (Lesslauer W. et al. Eur. J. Immunol., 1991, vol. 21, p. 2883; Ashkenazi A. et. al. PNAS USA, 1991, vol. 88, p. 10535; Peppel K. et al. J. Exp. Med., 1991, vol. 174, p. 1483; Mohler K. M. et al. J. Immunol. 1993, vol. 151, p. 1548).
With respect to the inhibition of TNF action or the control of immune responses using an Ig fusion protein, a multivalent or multimerized form of the extracellular domain as a functional domain of TNF receptors, CD2 and CTLA4 in an Ig fusion construct is expected to improve the efficacy of the fusion construct. When a monomeric fusion protein (heavy chain fusion protein) of the TNF receptor extracellular domain and the Ig heavy chain is expressed in a cell line simultaneously with another monomeric fusion protein (light chain fusion protein) of the TNF receptor extracellular domain and the Ig light chain, a dimeric fusion protein is produced by the interaction between heavy chain and the light chain. The dimeric fusion protein has two effective domains arranged in parallel like the in vivo form, and has remarkably increased efficacy in comparison with monomeric fusion constructs (Scallon B. J. et al. Cytokine, 1995, vol. 7, p. 759).
However, such an Ig fusion protein in a dimeric form is difficult to industrialize due to the following problems: two genes which are individually fused to the Ig heavy and light chains should be co-introduced into a host cell; when two different fusion proteins are simultaneously expressed in a single cell, their yields greatly decrease; and because all expressed heavy chain fusion proteins and light chain fusion proteins do not participate in the formation of dimers, dimers that fuse a heavy chain fusion protein and a light chain fusion protien are technically difficult to isolate from a mixture with the monomeric heavy chain fusion proteins or light chain fusion proteins.
In this regard, the present inventors constructed a concatameric protein in which a C-terminus of the soluble domain of a biologically active protein is linked to an N-terminus of the soluble domain of an identical or different biologically active protein, using recombinant DNA technology. Also, the present inventors prepared a DNA construct encoding a dimeric protein in which two molecules of a monomeric protein, in which a concatamer of a protein participating in an immune response is linked to the hinge region of an immunoglobulin Fc fragment are disulfide-bonded at the hinge region, and produced a concatamer-linked dimeric fusion protein using recombinant DNA technology based on the DNA construct.
As described above, attempts have been made to improve the efficacy and preparation method of immunoglobulin fusion proteins, but almost all efforts have been unable to increase the stability of the immunoglobulin fusion proteins. In this regard, as disclosed in Korean Pat. Application No. 2002-0045921, the present inventors developed a method of increasing protein stability by adding a glycosylation motif to a conjunction region between a functional domain of a protein and an immunoglobulin Fc region. However, when an immunoadhesin is glycosylated near a functional domain, the protein is not folded correctly or has reduced function.